(19)
Europaisches Patentamt
European Patent Office
Office europeen des brevets
(11)
EP 1 424 086 A1
(12)
EUROPEAN PATENT APPLICATION
(43)
Date of publication:
(51)
Intel 7: A61L 15/28
09 06 9004 Rullptin P004/P*?
(21)
Application number: 03254088.2
(22)
Date of filing: 27.06.2003
(84)
Designated Contracting States:
(72)
Inventors:
AT BE BG CH CY CZ DE DK EE ES Fl FR GB GR
•
Pendharkar, Sanyog Manohar
HU IE IT LI LU MC NL PT RO SE SI SK TR
Old Bridge, New Jersey 08857 (US)
Designated Extension States:
•
Gorman, Anne Jessica
AL LT LV MK
Highstown, NJ 08520 (US)
(30)
Priority: 26.11.2002 US 304472
(74)
Representative: Mercer, Christopher Paul et al
Carpmaels & Ransford
(71)
Applicant: ETHICON, INC.
43, Bloomsbury Square
Somerville, NJ 08876 (US)
London WC1A2RA (GB)
(54) Hemostatic wound dressing containing aldehyde-modified polysaccharide and hemostatic
agents
(57) The present invention is directed to hemostatic
wound dressings that contain a substrate for contacting
a wound, wherein the substrate includes a wound-con-
tacting surface and is fabricated at least in part from a
biocompatible aldehyde-modified polysaccharide hav-
ing covalently conjugated there with a hemostatic agent,
and to methods of providing hemostasis to a wound that
include applying the wound dressing described herein
to a wound.
Printed by Jouve, 75001 PARIS (FR)
EP 1 424 086 A1
Description
FIELD OF THE INVENTION
5 [0001 ] The present invention relates to hemostatic wound dressings containing or fabricated from an aldehyde-mod-
ified polysaccharide, e.g. aldehyde-modified regenerated cellulose, having covalently conjugated there with a hemo-
static agent, and to a method of providing hemostasis to a wound.
BACKGROUND OF THE INVENTION
10
[0002] The control of bleeding is essential and critical in surgical procedures to minimize blood loss, to reduce post-
surgical complications, and to shorten the duration of the surgery in the operating room. Oxidized cellulose, due to its
biodegradable, bactericidal, and hemostatic properties, has long been used as a topical hemostatic wound dressing
in a variety of surgical procedures, including neurosurgery, abdominal surgery, cardiovascular surgery, thoracic surgery,
15 head and neck surgery, pelvic surgery, and skin and subcutaneous tissue procedures.
[0003] The use of oxidized cellulose as a hemostat was first described by Virginia Franz in 1 944. Currently available
oxidized cellulose hemostats are knitted or non-woven fabrics comprising carboxylic oxidized cellulose. Oxidized re-
generated cellulose (ORC) is carboxylic-oxidized cellulose comprising reactive carboxylic acid groups. Examples of
ORC absorbable hemostats commercially available include Surgicel® absorbable hemostat, a knitted fabric of ORC;
20 Surgicel Nu-Knit® absorbable hemostat, a dense ORC fabric; and Surgicel® Fibrillar absorbable hemostat; all available
from Johnson & Johnson Wound Management Worldwide, a division of Ethicon, Inc., Somerville, New Jersey, a John-
son & Johnson Company. Other examples of commercial absorbable hemostats containing oxidized cellulose include
Oxycel® absorbable cellulose surgical dressing from Becton Dickinson and company, Morris Plains, New Jersey.
[0004] Although the absorbency of body fluid and the hemostatic action of currently available oxidized cellulose
25 hemostats are adequate for applications where mild to moderate bleeding is encountered, they are not known to be
effective to prevent or stop severe bleeding of high volume and high blood flow rate where a relatively high volume of
blood is lost at a relatively high rate, nor are they known to achieve rapid hemostasis. In such instances, e.g. arterial
puncture, liver resection, blunt liver trauma, blunt spleen trauma, aortic aneurysm, bleeding from patients with over-
anticoagulation, or patients with coagulopathies, such as hemophilia, etc., a higher degree of hemostasis is required
30 quickly.
[0005] In an effort to achieve enhanced hemostatic properties, blood-clotting agents, such as thrombin, fibrin and
fibrinogen have been combined with carriers or substrates. Aqueous solution of thrombin is routinely used with gelatin-
based carriers to enhance hemostasis at a surgical wound site. Two component fibrin sealants, consisting of thrombin
and fibrinogen/Factor XIII have been used as surgical hemostats in liquid form or as a solid patch in combination with
35 collagen matrix.
[0006] Physiologically, coagulation represents the transformation of soluble fibrinogen into an insoluble fibrin network
under the influence of thrombin, the key enzyme. During the normal clotting cascade, fibrinogen is cleaved by thrombin
and forms fibrin that polymerizes to form a fibrin clot, which is further strengthened by cross-linking by Factor XIII. Use
of fibrin sealants to a bleeding surface results in accelerated hemostasis and a sealing effect on the bleeding surface.
40 [0007] Thrombin is a coagulation factor associated with an extraordinary range of biological activities. Thrombin has
direct effects on coagulation, such as activating platelets, forming fibrin, and activating various procofactors and pro-
enzymes in the coagulation cascade. Its biological activity extends through anticoagulation, stimulation of fibrinolytic
reactions, activation of peripheral blood cell populations, and regulation of vascular tone. In addition to initiating proc-
esses leading to the sealing of a wound, thrombin is also responsible, in its role as a growth factor, in stimulating repair
45 to tissue damage associated with the wound itself.
[0008] Sakamoto et al. in JP60087225 describe immobilizing thrombin and Factor XIII on oxidized cellulose substrate
through a dehydrating condensation reaction, again using the acid oxidation product of cellulose as a substrate. How-
ever, the acidic nature of carboxylic oxidized cellulose substrate could rapidly denature and inactivate acid sensitive
proteins, including thrombin or fibrinogen, on contact. Much of the enzymatic activity of thrombin and Factor XIII could
50 be lost during the reaction. This makes it difficult to use the carboxylic-oxidized cellulose as a carrier for thrombin,
fibrinogen, fibrin, or other acid sensitive biologies and pharmaceutical agents.
[0009] Hemostatic wound dressings containing neutralized carboxylic-oxidized cellulose and protein based-hemo-
static agents, such as thrombin, fibrinogen and fibrin are known. Neutralized carboxylic-oxidized cellulosic materials
are prepared by treating the acidic carboxylic-oxidized cellulose with a water or alcohol solution of a basic salt of a
55 weak organic acid to elevate the pH of the cellulosic material to between 5 and 8 by neutralizing the acid groups on
the cellulose prior to addition of thrombin in order to make it thrombin compatible. A thrombin hemostatic patch was
disclosed, wherein thrombin was added to an acidic carboxylic oxidized regenerated cellulose or other material in
presence of an acid neutralizing agent, epsilon aminocaproic acid (EACA), to raise the pH of the material to a region
2
EP 1 424 086 A1
where thrombin can perform as a hemostat. While such neutralized carboxylic-oxidized cellulose may be thrombin
compatible, it is no longer bactericidal, because the anti-microbial activity of oxidized cellulose is due to its acidic nature.
[0010] Hemostatic agents such as thrombin, fibrinogen or fibrin, if not covalently combined with the substrate, may
be rinsed away by blood at a wound site. Alternatively, the non-bonded free form of thrombin, fibrinogen or fibrin, may
5 migrate into the blood stream and potentially cause severe thrombosis in procedures such as arterial puncture, liver
resection, blunt liver trauma, blunt spleen trauma, aortic aneurysm, etc., where higher blood pressure and higher blood
velocity is encountered. Therefore, caution must be taken to prevent thrombin from migrating to the blood stream.
[0011] The use of cotton gauze that has been modified by oxidation to contain aldehyde, and then further by car-
boxymethylation, sulfonation or phosphorylation, has been disclosed for use in wound dressings. However, such dress-
10 ings are not hemostatic and contain functional groups such as carboxymethyl, sulfonyl or phosphonyl groups.
[0012] Methods of producing highly oxidized tri-carboxylic acid derivatives of cellulose as hemostatic materials, in-
volving two-stage oxidation by successive processing with an iodine-containing compound and nitrogen oxides, has
been disclosed in RU21 46264 and IN1 59322. As disclosed in these disclosures, oxidized cellulosic materials were
prepared by preliminary oxidation with metaperiodate or periodic acid to yield periodate-oxidized, dialdehyde cellulose
15 to form the intermediate for forming OC. The dialdehyde cellulose intermediate then is further oxidized by N0 2 to yield
the OC, which is suitable for use as a hemostatic, anti-microbial and wound healing agent. The disclosures do not,
however, suggest or disclose that the periodate-oxidized, dialdehyde cellulose intermediate formed in the first stage
oxidation may or should be used in the preparation of wound dressings, e.g. hemostatic wound dressings.
[0013] To date, however, aldehyde-modified cellulose has not been utilized in wound dressings to provide hemos-
20 tasis. No method is taught in the prior art whereby a di-hydroxyl containing material such as cellulose is oxidized with
periodate to form an aldehyde-modified regenerated cellulose substrate. Nor has it been taught to covalently conjugate
an active hemostatic protein such as thrombin, fibrinogen or fibrin, with an aldehyde-modified regenerated cellulose
substrate to create a hemostatic device.
[0014] It would be advantageous to provide an anti-microbial hemostatic wound dressing that not only exhibits im-
25 proved hemostasis via the inclusion of hemostatic agents, such as thrombin, fibrinogen or fibrin, but does so without
the risk of the hemostatic agents migrating into the blood stream where they could cause severe thrombosis.
SUMMARY OF THE INVENTION
30 [0015] The present invention is directed to hemostatic wound dressings that contain a substrate for contacting a
wound, wherein the substrate comprises a wound-contacting surface and is fabricated at least in part from a biocom-
patible aldehyde-modified polysaccharide; and the substrate further includes a hemostatic agent covalently conjugated
with the aldehyde-modified polysaccharide. The invention also is directed to methods of providing hemostasis to a
wound that includes applying the wound dressing described herein to a wound.
35
DETAILED DESCRIPTION OF THE INVENTION
[0016] The present invention is directed to wound dressings comprising a biocompatible, hemostatic, wound con-
tacting and/or covering substrate comprising an aldehyde-modified polysaccharide having covalently conjugated there
40 with a hemostatic agent, for example, thrombin, fibrinogen or fibrin; and to methods of providing enhanced hemostasis
to wounds.
[0017] The hemostatic wound dressings of the present invention provide and maintain effective hemostasis when
applied to a wound requiring hemostasis. Effective hemostasis, as used herein, is the ability to control and/or abate
capillary, venous, or arteriole bleeding within an effective time, as recognized by those skilled in the art of hemostasis.
45 [0018] The hemostatic dressings of the present invention are particularly useful when conventional procedures to
control and/or abate bleeding, such as pressure or suturing, are either ineffective or impractical. The hemostatic wound
covering substrates of the present invention comprise covalently conjugated there with hemostatic agents, or other
biological or therapeutic compounds, moieties or species, particularly those "acid-sensitive" agents that may be de-
graded or denatured by, or otherwise detrimentally affected by acidic pH such as is provided by conventional OC
50 hemostats.
[0019] The wound dressings may take various physical forms and may include, without limitation, fibrous or non-
fibrous, knitted, woven or non-woven dressings. In preferred embodiments, the wound dressing may comprise a fiber,
including microfibers, a film, a fabric, a foam, a bead, a powder, a gel, or combinations thereof. Regardless of the form
of the wound dressing, it will comprise a substrate forcontacting and/or covering the wound. In certain wound dressings,
55 the dressing may consist essentially of the substrate, or may consist of the substrate. This is particularly true where
the wound dressing is fabricated from a knitted, woven or non-woven hemostatic fabric that has been oxidized to
provide aldehyde modification, as described herein, and which serves as the substrate for the wound dressing. In those
cases, while the wound dressing may further include such components as backing layers, adhesive layers, or the like,
3
EP 1 424 086 A1
the wound dressing can include only the hemostatic fabric.
[0020] The wound dressing substrate will comprise a wound-contacting surface. Such substrates may take various
physical forms, including, but not limited to, fibrous or non-fibrous, knitted, woven or non-woven substrates. In certain
embodiments, the wound dressing substrates may comprise a fiber, including microfibers, a film, a fabric, a foam, a
bead, a powder, a gel, or combinations thereof. In preferred embodiments, the substrate comprises a knitted or a woven
fabric. The fabric may be formed, cut or otherwise shaped to cover the wound surface, thereby providing protection of
the wound from physical trauma and effective hemostasis of the wound.
[0021] Wound dressings of the present invention, and more particularly the wound-contacting substrates thereof,
comprise a biocompatible, aldehyde-modified polysaccharide. In preferred wound dressings, the polysaccharide will
contain an amount of aldehyde moieties effective to render the modified polysaccharide biodegradable, meaning that
the polysaccharide is degradable by the body into components that either are resorbable by the body, or that can be
passed readily by the body. More particularly, the biodegraded components do not elicit permanent chronic foreign
body reaction because they are absorbed by the body, such that no permanent trace or residual of the component is
retained at the implantation site.
[0022] Aldehyde-modified polysaccharides used in the present invention may be prepared from biocompatible
polysaccharides that are useful in medical devices. Such polysaccharides include, without limitation, cellulose, alkyl
cellulose, e.g. methyl cellulose, hydroxyalkyl cellulose, alkylhydroxyalkyl cellulose, cellulose sulfate, salts of car-
boxymethyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, chitin, carboxymethyl chitin, hyaluronic acid,
salts of hyaluronic acid, alginate, alginic acid, propylene glycol alginate, glycogen, dextran, dextran sulfate, curdlan,
pectin, pullulan, xanthan, chondroitin, chondroitin sulfates, carboxymethyl dextran, carboxymethyl chitosan, chitosan,
heparin, heparin sulfate, heparin sulfate, dermatan sulfate, keratin sulfate, carrageenans, chitosan, starch, amylose,
amylopectin, poly-N-glucosamine, polymannuronic acid, polyglucuronic acid, polyguluronic acid, and derivatives of
any of the above. In preferred embodiments, the polysaccharide is oxidized as described herein to assure that the
aldehyde-modified polysaccharide is biodegradable.
[0023] Such biodegrable, aldehyde-modified, regenerated polysaccharides may be represented by Structure I below.
x+y = 100%
where x and y represent mole percent, x plus y equals 100 percent, x is from about 95 to about 5,
y is from about 5 to about 95; and
R may be CH 2 OR 3 , , COOR 4 , sulphonic acid, or phosphonic acid; R 3 and R 4 may be H, alkyl, aryl, alkoxy or aryloxy,
and R 1 and R 2 may be H, alkyl, aryl, alkoxy, aryloxy, sulphonyl or phosphoryl.
[0024] In preferred embodiments of the present invention, the biocompatible, biodegradable hemostatic wound
dressing comprises a wound contacting/covering substrate prepared from a biocompatible, biodegradable, aldehyde-
modified, regenerated polysaccharide. Regenerated cellulose is preferred due to its higher degree of uniformity versus
cellulose that has not been regenerated. Regenerated cellulose is described in, for instance, United States Patent
3,364,200, the contents of which is hereby incorporated by reference as if set forth in its entirety.
[0025] In particular, preferred aldehyde-modified regenerated cellulose is one comprising repeating units of Structure
II below:
EP 1 424 086 A1
10
x+y = 1 00%
15
where x and y represent mole percent, x plus y equals 100 percent, x is from about 95 to about 5,
y is from about 5 to about 95; and R is CH 2 OH, R 1 and R 2 are H.
[0026] In certain embodiments according to the present invention, x is from about 90 to 1 0 and y is about 1 0 to about
20 90. Preferably, x is from about 80 to 20 and y is from about 20 to about 80. Even more preferably, x is from about 70
to about 30. Most preferably, x is about 70 and y is about 30.
[0027] The hemostatic dressings of the present invention also provide anti-microbial activities due to the presence
of effective amounts of the aldehyde moieties. It has been shown that in spite of being non-acidic, the aldehyde-modified
regenerated cellulose is anti-microbial in nature. The hemostats of the present invention were found to be significantly
25 effective against microorganisms, such as Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas
aeruginosa, etc. The anti-microbial activities of the non-acidic aldehyde-modified regenerated cellulose are shown to
be comparable to those of the acidic carboxylic oxidized regenerated cellulose conventionally used. The acidic car-
boxylic oxidized regenerated cellulose loses its anti-microbial activities upon neutralization reaction or over a period
of time as the acid groups are neutralized in the body. However, the aldehyde-modified regenerated cellulose utilized
30 in the present invention is expected to retain its anti-microbial activity over a longer period of time.
[0028] In preferred embodiments of the invention, the aldehyde-modified regenerated polysaccharide is essentially
free of functional or reactive moieties other than aldehyde moieties. By essentially free, it is meant that the polysac-
charide does not contain such functional or reactive moieties in amounts effective to alter the properties of the aldehyde-
modified polysaccharide or to provide the substrate comprising the polysaccharide with a pH of less than about 4.5,
35 more preferably less than about 5, or greater than about 9, preferably about 9.5. Such moieties include, without limi-
tation, carboxylic acid moieties typically present on wound dressings made from OC. Excess levels of carboxylic acid
moieties will lower the pH of the substrates and dressings so that they are not compatible for use with those acid
sensitive species that may be degraded or denatured by such a low pH, e.g. thrombin. Other moieties include, without
limitation, sulfonyl or phosphonyl moieties.
40 [0029] The hemostat of the present invention exhibits increased thermal stability compared to that of the carboxylic
oxidized regenerated cellulose fabric (ORC). The increased thermal stability may be indicative of improved physical
shelf-life, compared to ORC or neutralized ORC.
[0030] In certain embodiments of the invention, the fabrics utilized in the present invention may be knitted, woven
or non-woven, provided that the fabric possesses the physical properties adequate for wound dressings, in general,
45 and hemostatic wound dressings, specifically. Fabrics oxidized by periodic acid or its salts described in the present
invention are expected to retain physical properties and mechanical integrity required for use in wound dressings.
Hemostatic fabrics useful for use in hemostatic wound dressings according to the present invention include fabrics
comprising the aldehyde-modified polysaccharides of the present invention and being of the structure described in
United States Patent Numbers 2,773,000, 3,364,200, 4,626,253, and 5,002,551 , the contents each of which is hereby
50 incorporated by reference herein as if set forth in its entirety.
[0031] In certain embodiments of the invention, the hemostatic wound dressing of the present invention comprises
as the wound contacting/covering hemostatic substrate a warp knitted tricot fabric constructed of bright rayon yarn that
has been oxidized by periodic acid or its salts such that the substrate comprises aldehyde moieties. Both Scanning
Electron Microscopic (SEM) images and fabric mechanical properties indicate that the physical characteristics (density,
55 thickness) and physical performance, e.g. fabric tensile strength and Mullen burst strength, of the aldehyde-modified
regenerated cellulose in the present invention are comparable to those of the fabric disclosed in United States Patent
4,626,253.
[0032] The hemostat of the present invention remains very flexible, conforms to a bleeding site, and retains good
5
EP 1 424 086 A1
tensile and compressive strength to withstand handling during application. The aldehyde-modified regenerated cellu-
lose hemostat can be cut into different sizes and shapes to fit the surgical needs. It can be rolled up or packed into
irregular anatomic areas.
[0033] Other warp knit tricot fabric constructions which produce equivalent physical properties may, of course, be
5 utilized in the manufacture of the aldehyde-modified regenerated cellulose hemostatic wound dressings of the present
invention, and such constructions will be apparent to those skilled in the art once having the benefit of this disclosure.
[0034] In other embodiments, the hemostat of the present invention comprises of powdered or pulverized aldehyde-
modified regenerated cellulose fabric conjugated with the hemostatic agents.
[0035] In certain embodiments of the invention, a biologies, a drug or a combination of pharmaceutical agents that
10 otherwise may be sensitive to the low pH of OC-containing wound dressings, such agents may be incorporated into
certain wound dressings of the present invention without having to adjust pH prior to incorporation into the dressing.
To fabricate such a hemostatic wound dressing, a drug or agent is first dissolved in an appropriate solvent. The fabric
is then coated with the drug solution, and the solvent is removed. Preferred biologies, drugs and agent include anal-
gesics, anti-infective agents, antibiotics, adhesion preventive agents, pro-coagulants, and wound healing growth fac-
15 tors.
[0036] The aldehyde groups formed on the polysaccharide matrix during the periodate oxidation reaction can be
used to covalently bond amine containing biologies and therapeutic agents. The combination of such biologies, drugs
and agents with wound dressings of the present invention using the aldehyde-modified regenerated cellulose sub-
strates can provide improved hemostatic wound dressings, wound healing dressings, drug delivery devices, and tissue
20 engineering matrices.
[0037] Substrates used in wound dressings of the present invention comprise an aldehyde-modified polysaccharide
comprising covalently conjugated there with a hemostatic agent bearing an aldehyde reactive moiety. The hemostatic
agent, including procoagulant enzymes, proteins and peptides, can be naturally occurring, recombinant, or synthetic,
and may be selected from the group consisting of prothrombin, thrombin, fibrinogen, fibrin, fibronectin, heparinase,
25 Factor X/Xa, Factor VI l/VI la, Factor IX/IXa, Factor Xl/XIa, Factor XII/XI la, tissue factor, batroxobin, ancrod, ecarin, von
Willebrand Factor, collagen, elastin, albumin, gelatin, platelet surface glycoproteins, vasopressin and vasopressin ana-
logs, epinephrine, selectin, procoagulant venom, plasminogen activator inhibitor, platelet activating agents, synthetic
peptides having hemostatic activity, and any combination thereof. Preferred hemostatic agents in the present invention
are thrombin, fibrinogen and fibrin.
30 [0038] The aldehyde moiety of aldehyde-modified regenerated polysaccharide can readily react with the amine
groups present on the amino acid side chains or N-terminal residues of thrombin, fibrinogen or fibrin, resulting in forming
a conjugate of the hemostatic agent with the aldehyde-modified regenerated polysaccharide covalently linked by a
reversible imine bond. The imine bonded aldehyde-modified regenerated polysaccharide/hemostatic agent conjugate
may then be further reacted with a reducing agent such as sodium borohydride or sodium cyanoborohydride to form
35 an irreversible secondary amine linkage. In preferred embodiments of the invention, the hemostatic agent is dispersed
at least on the wound-contacting surface of the substrate, and preferably at least partially through the wound contacting
substrate, bonded covalently to the aldehyde-modified polysaccharide by reversible or irreversible bonds.
[0039] Oxidation of 2, 3- vicinal hydroxyl groups in a carbohydrate with periodic acid (or any alkali metal salt thereof)
forms a di-aldehyde or di-aldehyde derivatives. These aldehyde moieties(-RCH(0)) can then readily react with a pri-
40 mary amine moiety (-NH 2 ), such as are present on the amino acid side chains or N-terminal residues of proteins,
resulting in an equilibrium with the reaction product, a protein and carbohydrate conjugate, covalently linked by a
relatively unstable and reversible imine moiety (-N=CHR). To stabilize the linkage between the biomolecule and the
substrate surface, subsequent reductive alkylation of the imine moiety is carried out using reducing agents (i.e., sta-
bilizing agents) such as, for example, sodium borohydride, sodium cyanoborohydride, and amine boranes, to form a
45 secondary amine (-NH-CH 2 -R) .
[0040] As noted above, wound dressings of the present invention provide rapid hemostasis and maintain effective
hemostasis in cases of severe bleeding. Examples of severe bleeding include, without limitation, arterial puncture, liver
resection, blunt liver trauma, blunt spleen trauma, aortic aneurysm, bleeding from patients with over-anticoagulation,
or bleeding from patients with coagulopathies, such as hemophilia. Protein based hemostatic agents, such as thrombin,
50 fibrin or fibrinogen, if covalently conjugated to the aldehyde groups of the aldehyde-modified polysaccharide to form
a secondary amine linkage by converting the imine bond with reducing agents such as sodium borohydride or sodium
cyanoborohydride bond, can enhance the hemostatic property of aldehyde-modified regenerated cellulose wound
dressings and reduce the risk of thrombosis caused by free hemostatic agents migrating into the blood stream.
[0041] The hemostatic wound dressing of the present invention comprises hemostatic agents, including but not lim-
55 ited to thrombin, fibrinogen or fibrin, in an amount effective to provide rapid hemostasis and maintain effective hemos-
tasis in cases of severe bleeding. If the concentration of the hemostatic agent on the aldehyde-modified regenerated
cellulose substrate is too low, the hemostatic agents do not provide an effective proagulant activity to promote rapid
clot formation upon contact with blood or blood plasma. A preferred concentration range of thrombin on aldehyde-
6
EP 1 424 086 A1
modified regenerated cellulose substrate is from about 0.001 to about 1 percent by weight. A more preferred concen-
tration of thrombin on aldehyde-modified regenerated cellulose substrate is from about 0.01 to about 0.1 percent by
weight. A preferred concentration range of fibrinogen on the aldehyde-modified regenerated cellulose substrate is from
about 0.1 to about 50 percent by weight. A more preferred concentration of fibrinogen on the aldehyde-modified re-
5 generated cellulose substrate is from about 2.5 to about 1 0 by weight. A preferred concentration range of fibrin on the
aldehyde-modified regenerated cellulose substrate is from about 0.1 to about 50 percent by weight. A more preferred
concentration of fibrin on the aldehyde-modified regenerated cellulose substrate is from about 2.5 to about 1 0 by weight.
[0042] The features of such covalently bonded hemostatic agents conjugated with the aldehyde-modified regener-
ated cellulose wound dressing can be controlled to suit a desired application by choosing the conditions to form the
10 composite hemostat during conjugation.
[0043] In certain embodiments of the present invention, the hemostatic agent, such as thrombin, fibrinogen or fibrin,
is dispersed substantially homogeneously through the wound dressing substrate. In such cases, aldehyde-modified
regenerated cellulose substrate may be immersed in the solution of thrombin, fibrinogen or fibrin to provide homoge-
neous distribution throughout the wound dressing.
15 [0044] In other embodiments of the present invention, a faster hemostat can be created by the following procedure.
The aldehyde-modified regenerated cellulose wound dressing can be soaked with the desired amount of aqueous
solution of thrombin and rapidly lyophilized using known methods that retain therapeutic activity. The dry hemostatic
biologic conjugate can be used as a fast hemostat with excellent bactericidal activity, biodegradability, bioabsorbability
and long-lasting stability.
20 [0045] In other embodiments, it is preferred that aldehyde-modified regenerated cellulose substrate is soaked with
a solution of fibrinogen and subsequently exposed to thrombin prior to lyophilization.
[0046] In certain embodiments of the invention, the thrombin conjugate of aldehyde-modified regenerated cellulose
substrate is further reacted with reducing agents such as sodium borohydride or sodium cyanoborohydride to form a
secondary amine linkage. The aldehyde-modified regenerated cellulose substrate can be soaked with the desired
25 amount of aqueous solution of thrombin, then reacted with aqueous solution of sodium borohydride or sodium cy-
anoborohydride reconstituted in phosphate buffer (PH=8) prior to lyophilization.
[0047] The reduced form of the aldehyde-modified regenerated cellulose-thrombin conjugate is more stable due to
the nature of the secondary amine linkage. Hemostatic wound dressings of this embodiment have enhanced hemostatic
properties, as well as increased stability, and can provide rapid hemostasis without causing thrombin to migrate into
30 the blood stream and cause severe thrombosis.
[0048] In other embodiments of the present invention, it is preferred thatthrombin is constituted in an aqueous solution
of a non-acidic water-soluble polymer, including but not limited to alkyl cellulose, e.g. methyl cellulose, hydroxyalkyl
cellulose, alkyl hydroxyalkyl cellulose, salts of carboxymethyl or carboxyethyl cellulose, chitin, salts of hyaluronic acid,
alginate, propylene glycol alginate, glycogen, dextran, carrageenans, chitosan, starch, amylose, and poly-N-glu-
35 cosamine. The aldehyde-modified regenerated cellulose wound dressing can be soaked with the desired amount of
aqueous solution of thrombin and the water-soluble polymer and rapidly lyophilized using known methods that retain
therapeutic activity. The dry hemostatic biologic conjugate patch can be used as a fast hemostat.
[0049] In certain embodiments of the invention, a biologic, a drug or a combination of pharmaceutical agents can be
incorporated into the hemostat without adjusting it pH value. Preferred agents include but not limited to analgesics,
40 anti-infective agents, antibiotics, adhesion preventive agents, procoagulants, and wound healing growth factors. To
construct such a hemostat, a pharmaceutical agent is first dissolved in an appropriate solvent. The wound dressing is
then coated with such solution, and the solvent is removed. The combination of such biologies, drugs and agents with
the aldehyde-modified oxidized regenerated cellulose hemostat of the present invention can construct faster hemostat,
better wound healing device, drug delivery device, and tissue engineering matrix.
45 [0050] While the following examples demonstrate certain embodiments of the invention, they are not to be interpreted
as limiting the scope of the invention, but rather as contributing to a complete description of the invention. Treatment
times and temperatures for reactions in the examples below tend to be inversely related. Higher temperatures require
relatively shorter treatment times. The limitations of the time and temperature are governed by the effect on the bio-
logical stability of the hemostatic agents. Conditions outside what is described below are still within the scope of this
50 invention.
Example 1 :
Preparation of knitted aldehyde-modified regenerated cellulose fabric:
55
[0051] A 15.75 g piece of Nu-Knit® rayon fabric was cut in the form of a strip 1 .5 inches wide. The strip was wound
on a mandrel and suspended in 600 ml of aqueous isopropyl alcohol (IPA) (200 ml IPA/400 ml de-ionized (Dl) water).
20.8 g of sodium periodate (Aldrich, Milwaukee, 53201 ) was dissolved in the solution (1 :1 molar ratio) and the mandrel
7
EP 1 424 086 A1
was rotated at moderate rpm in the solution for 21 hours at ambient temperature. It is essential that the oxidation of
the fabric be conducted in the dark. The solution pH was 3.8. The solution was discarded after the reaction. The mandrel
with the oxidized fabric was washed for 30 minutes in 1 liter of cold Dl water containing 50 ml of ethylene glycol. It was
then washed with aqueous IPA (50/50) for 15 minutes, followed by a pure IPA wash for 15 minutes. The fabric was
5 dried in ambient air for several hours. [Aldehyde content: Ave. 22.83%]
[0052] The oxidized fabric then was evaluated for hemostasis as set forth below. Results are provided in Table 1 .
Example 2:
10 Preparation of non-woven aldehyde-modified cellulose fabric:
[0053] A 1 0 g piece of cellulose rayon non-woven fabric was cut in the form of a rectangle and placed in an aqueous
solution of sodium periodate (Aldrich, Milwaukee, 53201) (1:0.7 molar ratio). The fabric was placed in a container
modified to exclude light and soaked in the dark for 24 hours at 37°C. The solution was discarded after the reaction.
15 The fabric was repeatedly washed with Dl water until the pH was 6-7. It was then washed with aqueous IPA (50/50)
for 1 5 minutes. The fabric then was washed in pure I PA for 1 5 minutes. The fabric was dried in ambient air for several
hours, [aldehyde content: 51.04%]
[0054] The oxidized fabric then was evaluated for hemostasis as set forth below. Results are provided in Table 1 .
20 Example 3:
Preparation of aldehyde-modified regenerated cellulose powders:
[0055] 10.6 g of powdered cellulose rayon was suspended in an aqueous solution of sodium periodate (Aldrich,
25 Milwaukee, 53201 )(1 3.9 g in 250 ml Dl water] and stirred for 7 hours at ambient temperature in the dark. The solution
was filtered after the reaction. The filtrate was repeatedly washed with Dl water until the pH was in the range of from
6 to 7. It was then washed with aqueous IPA (50/50) and pure IPA for 15 min each. The powder was dried in air for
several hours, [aldehyde content: 32.8 %]
[0056] The oxidized powder then was evaluated for hemostasis as set forth below. Results are provided in Table 1 .
30
Example 4:
Preparation of aldehyde-modified cellulose beads:
35 [0057] 13.67 g of porous cellulose beads are floated in an aqueous solution of sodium periodate (Aldrich, Milwaukee,
53201) (18g in 250 ml Dl water/1 25ml IPA) and stirred for 24 hours at ambient temperature. The material was filtered
and the filtrate (beads and crushed beads) was repeatedly washed with Dl water until the pH was in the range of from
6 to 7. It was then washed with aqueous IPA (50/50) and pure IPA for 15 min each. The material was dried in air for
several hours, [aldehyde content: intact beads-29.86 %; crushed beads-35%]
40 [0058] Thrombin conjugates with the oxidized beads were prepared similar to methods disclosed herein. The oxidized
beads and thrombin conjugates then were evaluated for hemostasis as set forth below. Results are provided in Table 1 .
Example 5:
45 Thrombin conjugated with aldehyde-modified regenerated cellulose
[0059] An 8 g piece of fabric prepared in Example 1 was soaked in 20 ml of freshly reconstituted thrombin solution
(1 000 units/ml) in a flat metal pan. The thrombin solution accordingly was distributed throughout the fabric substrate.
The pan was quickly introduced into a pre-cooled freezer maintained at -20°C. The material was stored frozen. The
50 pan was transferred into a " Virtis Advantage" lyophilizer with a shelf-temperature of -50°C. The pan was maintained
at that temperature under vacuum for 6 hours. The temperature was raised and maintained at -15°C for another 2
hours. It was then subsequently raised to 0°C and 15°C for 16 hours at each temperature. At this time the water had
completely sublimed. The vacuum was released and the fabric was removed from the pan. The thrombin, covalently
conjugated with the aldehyde-modified regenerated cellulose, was distributed throughout the substrate via the lyophi-
55 lization of the fabric in solution. The flexible material was stored in the refrigerator in an airtight container until further
use. A portion of the lyophilized fabric conjugate was pulverized into a powder.
[0060] The thrombin-conjugated aldehyde-modified regenerated cellulose fabric then were evaluated for hemostasis
as set forth below. Results are provided in Table 1 .
8
EP 1 424 086 A1
Example 6:
Thrombin conjugated with aldehyde-modified regenerated cellulose and immobilized by reduction.
5 [0061] A 3.2 g piece of fabric prepared according to Example 1 was soaked in 8 ml of thrombin solution in phosphate
buffer (pH = 8) at 800 units/ml in a flat metal pan ('A'). In another pan ('B'), 2.9 g of the same fabric was similarly soaked
with 8 ml of the thrombin solution. Both pans were quickly introduced into a pre-cooled freezer maintained at -20°C.
After 13 hours, pan 'A' was thawed and the wet fabric was quickly transferred into a large centrifuge tube containing
45 ml of (50 mM) NaCNBH 4 reconstituted in phosphate buffer (pH 8). The fabric was completely submerged in the
10 solution for 1 5 min. The fabric was isolated and repeatedly washed with Dl water. The final wet fabric was placed on
the pan and frozen at -20°C. Both pans were quickly transferred into a 'Virtis Advantage' lyophilzer with a shelf-tem-
perature of -50°C. They were maintained at that temperature under vacuum for 2 hours. The temperature was raised
and maintained at -15°C for another 12 hours. It was then subsequently raised to 0°C and 15°C for 2 hours at each
temperature. At this time the water had completely sublimed. The vacuum was released and the fabrics were removed
15 from the pan. The flexible materials were stored in the refrigerator in an airtight container until further use.
Example 7:
Fibrinogen conjugated with aldehyde-modified regenerated Cellulose
20
[0062] An 8 g piece of fabric as produced according to Example 1 was soaked in 20 ml of freshly reconstituted
fibrinogen solution (40 mg/ml) in a flat metal pan. The fabric was lyophilized and, as before, and a portion pulverized
as in Example 5. The fabric was evaluated and was evaluated for hemostasis as set forth below. Results are provided
in Table 1 .
25
Example 8:
Fibrin conjugated with aldehyde-modified regenerated Cellulose
30 [0063] An 8 g piece of fabric according to Example 1 was soaked in 20 ml of freshly reconstituted fibrinogen solution
(40 mg/ml) in a flat metal pan. This was sprayed with an equal amount of thrombin solution (1 000 unit/ml). A gel was
rapidly formed. The pan was quickly introduced and stored in a pre-cooled freezer maintained at -20°C. The pan was
subsequently transferred into a 'Virtis Advantage' lyophilzer with a shelf-temperature of -50°C. The pan was maintained
at that temperature under vacuum for 2 hours. The temperature was raised and maintained at -15°C for another 12
35 hours. It was then subsequently raised to 0°C and 15°C for 2 hours at each temperature. At this time the water had
completely sublimed. The vacuum was released and the fabric was removed from the pan. The flexible material was
stored in the refrigerator in an airtight container under further use.
[0064] The fibrin conjugated aldehyde-modified regenerated cellulose fabric then was evaluated for hemostasis as
set forth below. Results are provided in Table 1 .
40
Example 9:
Blends of powder conjugates.
45 [0065] Pulverized conjugates as prepared in Examples 5 and 7 were blended and evaluated for hemostatis as set
forth below. Results are presented in Table 1 .
Example 10:
50 Hemostatic performance of different materials in porcine splenic incision model
[0066] A porcine spleen incision model was used for hemostasis evaluation of different materials. The materials were
cut into 2.5 cm X 2.0 cm rectangles. A linear incision of 1 .5 cm with a depth of 1 .0 cm was made with a surgical blade
on a porcine spleen. After application of the test article, digital tamponade was applied to the incision for 2 minutes.
55 The hemostasis was then evaluated. Additional applications of digital tamponade for 30 seconds each time were used
until complete hemostasis was achieved. Fabrics failing to provide hemostasis within 12 minutes were considered to
be failures. Wound dressings comprising aldehyde-modified regenerated cellulose achieve rapid hemostasis compared
to the negative control of surgical gauze, as shown in table 1. Observations on effectiveness of thrombin, fibrinogen
9
EP 1 424 086 A1
and fibrin as hemostatic agents in reducing time to hemostasis are also shown in table 1.
Table 1
Hemostatic performance of Aldehyde-Modified Regenerated Cellulose (AMRC) Based-Materials
Example No.
Sample
Time to Hemostasis (seconds)
1
AMRC knitted fabric
187 (n=11)
5
AMRC/Thrombin (fabric)
30 (n=3)
8
AMRC /fibrin (fabric)
30 (n=4)
7
AMRC/fibrinogen (fabric)
65 (n=2)
2
AMRC Non-woven fabric
96 (n=5)
3
AMRC powder
120 (n=3)
5
AMRC/Thrombin (powder)
30 (n=3)
8
AMRC/fibrin (powder)
30 (n=1)
9
AMRC/thrombin powder plus AMRC/fibrinogen powder
250 (n=1)
4
AMRC Beads
238 (n=1)
4
AMRC Beads/Thrombin
30 (n=3)
Surgical gauze Control
>720 (n=6)
Claims
1. A hemostatic wound dressing, comprising:
a substrate for contacting a wound, said substrate comprising,
a wound-contacting surface,
a biocompatible aldehyde-modified polysaccharide; and
a hemostatic agent covalently conjugated with said aldehyde-modified polysaccharide, said agent comprising
an aldehyde-reactive moiety,
wherein said wound dressing is hemostatic.
2. The wound dressing of claim 1 wherein said substrate comprises a fiber, a fabric, a sponge, a foam, a film, a bead,
a gel, a powder, or combinations thereof
3. The wound dressing of claim 1 wherein said aldehyde-modified polysaccharide is selected from the group con-
sisting of aldehyde-modified cellulose, alkyl cellulose, hydroxyalkyl cellulose, alkylhydroxyalkyl cellulose, cellulose
sulfate, salts of carboxymethyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, chitin, carboxymethyl
chitin, hyaluronic acid, salts of hyaluronic acid, alginate, alginic acid, propylene glycol alginate, glycogen, dextran,
dextran sulfate, curdlan, pectin, pullulan, xanthan, chondroitin, chondroitin sulfates, carboxymethyl dextran, car-
boxymethyl chitosan, chitosan, heparin, heparin sulfate, heparin sulfate, dermatan sulfate, keratin sulfate, carra-
geenans, chitosan, starch, amylose, amylopectin, poly-N-glucosamine, polymannuronic acid, polyglucuronic acid,
polyguluronic acid and derivatives of the above.
4. The wound dressing of claim 3 wherein said aldehyde-modified polysaccharide comprises an amount of aldehyde
effective to render the polysaccharide biodegradable.
5. The wound dressing of claim 4 wherein said aldehyde-modified polysaccharide is selected from the group con-
sisting of aldehyde-modified starch, dextran, pectin, alginate, chitin, chitosan, glycogen, amylose, amylopectin,
cellulose and cellulose derivatives.
6. The wound dressing of claim 5 wherein said aldehyde-modified polysaccaride comprises aldehyde-modified re-
generated polysaccharide.
10
EP 1 424 086 A1
7. The wound dressing of claim 6 wherein said aldehyde-modified polysaccharide comprises aldehyde-modified re-
generated cellulose comprising repeating units of structure II,
wherein x plus y equals 1 00 percent, x ranges from about 95 to about 5 percent, and
y ranges from about 5 to about 95 percent and R is CH 2 OH, and R 1 and R 2 are H.
8. The wound dressing of claim 7 wherein x ranges from about 80 to about 20 percent and y ranges from about 20
to about 80 percent.
9. The wound dressing of claim 8 wherein x is about 70 percent and y is about 30 percent.
10. The wound dressing of claim 1 wherein said aldehyde-modified polysaccharide is essentially free of carboxylicacid.
11. The wound dressing of claim 7 wherein said aldehyde-modified cellulose is essentially free of carboxylic acid.
12. The wound dressing of claim 1 wherein said hemostatic agent is synthetic, recombinant or naturally occurring.
13. The wound dressing of claim 1 wherein said hemostatic agent is selected from the group consisting prothrombin,
thrombin, fibrinogen, fibrin, fibronectin, heparinase, Factor X/Xa, Factor VI l/VI la, Factor IX/ 1 Xa, Factor Xl/XIa, Fac-
tor Xll/Xlla, tissue factor, batroxobin, ancrod, ecarin, von Willebrand Factor, collagen, elastin, albumin, gelatin,
platelet surface glycoproteins, vasopressin, vasopressin analogs, epinephrine, selectin, procoagulantvenom, plas-
minogen activator inhibitor, platelet activating agents and synthetic peptides having hemostatic activity.
14. The wound dressing of claim 1 wherein said substrate comprises from about 0.001 to about 50 percent by weight
of said hemostatic agent.
15. The wound dressing of claim 11 wherein said substrate comprises from about 0.001 to about 1 percent by weight
of thrombin as the hemostatic agent.
16. The wound dressing of claim 15 wherein said substrate comprises from about 0.01 to about 0.1 percent by weight
of thrombin as the hemostatic agent.
17. The wound dressing of claim 11 wherein said substrate comprises from about 0.1 to about 50 percent by weight
of fibrinogen as the hemostatic agent.
18. The wound dressing of claim 17 wherein said substrate comprises from about 2.5 to about 10 percent by weight
of fibrinogen as the hemostatic agent.
19. The wound dressing of claim 11 wherein the substrate comprises from about 0.1 to about 50 percent by weight of
fibrin as the hemostatic agent.
20. The wound dressing of claim 1 9 wherein the substrate comprises from about 2.5 to about 1 0 percent by weight of
fibrin as the hemostatic agent.
x+y = 1 00%
OR 2
- 1 y
X
11
EP 1 424 086 A1
21.
22.
5
23.
24.
10
15
20
25.
26.
25
30
27.
35
28.
29.
The wound dressing of claim 1 wherein said hemostatic agent is dispersed at least partially through said substrate.
The wound dressing of claim 1 wherein said hemostatic agent is conjugated with said aldehyde-modified polysac-
charide by covalent imine bonding.
The wound dressing of claim 1 wherein said hemostatic agent is conjugated with said aldehyde-modified polysac-
charide by covalent secondary amine linkage.
A method of providing hemostasis to a wound, comprising:
applying to a wound a hemostatic wound dressing, comprising:
a substrate for contacting a wound, said substrate comprising,
a wound-contacting surface,
a biocompatible aldehyde-modified polysaccharide; and
a hemostatic agent covalently conjugated with said aldehyde-modified polysaccharide, said agent com-
prising an aldehyde-reactive moiety,
wherein said wound dressing is hemostatic.
The method of claim 24 wherein said substrate comprises a fiber, a fabric, a sponge, a foam, a film, a bead, a gel,
a powder, or combinations thereof
The method of claim 24 wherein said aldehyde-modified polysaccharide is selected from the group consisting of
aldehyde-modified cellulose, alkyl cellulose, hydroxyalkyl cellulose, alkylhydroxyalkyl cellulose, cellulose sulfate,
salts of carboxymethyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, chitin, carboxymethyl chitin, hy-
aluronic acid, salts of hyaluronic acid, alginate, alginic acid, propylene glycol alginate, glycogen, dextran, dextran
sulfate, curdlan, pectin, pullulan, xanthan, chondroitin, chondroitin sulfates, carboxymethyl dextran, carboxymethyl
chitosan, chitosan, heparin, heparin sulfate, heparin sulfate, dermatan sulfate, keratin sulfate, carrageenans, chi-
tosan, starch, amylose, amylopectin, poly-N-glucosamine, polymannuronicacid, polyglucuronicacid, polyguluronic
acid and derivatives of the above.
The method of claim 26 wherein said aldehyde-modified polysaccharide comprises an amount of aldehyde effective
to render the polysaccharide biodegradable.
The method of claim 27 wherein said aldehyde-modified polysaccaride comprises aldehyde-modified regenerated
polysaccharide.
The wound dressing of claim 27 wherein said aldehyde-modified polysaccharide comprises aldehyde-modified
regenerated cellulose comprising repeating units of structure II,
O
O
R
\
O
x+y = 1 00%
OR 2
y
x
wherein x plus y equals 1 00 percent, x ranges from about 95 to about 5 percent, and
y ranges from about 5 to about 95 percent and R is CH 2 OH, and R 1 and R 2 are H.
12
EP 1 424 086 A1
30. The method of claim 29 wherein x ranges from about 80 to about 20 percent and y ranges from about 20 to about
80 percent.
31. The method of claim 24 wherein said aldehyde-modified polysaccharide is essentially free of carboxylic acid.
32. The method of claim 29 wherein said aldehyde-modified cellulose is essentially free of carboxylic acid.
33. The wound of claim 24 wherein said hemostatic agent is synthetic, recombinant or naturally occurring.
34. The methodof claim 33 wherein said hemostatic agent is selected from the group consisting prothrombin, thrombin,
fibrinogen, fibrin, fibronectin, heparinase, Factor X/Xa, Factor Vll/Vlla, Factor IX/IXa, Factor Xl/XIa, Factor XII/
XI la, tissue factor, batroxobin, ancrod, ecarin, von Willebrand Factor, collagen, elastin, albumin, gelatin, platelet
surface glycoproteins, vasopressin, vasopressin analogs, epinephrine, selectin, procoagulant venom, plasmino-
gen activator inhibitor, platelet activating agents and synthetic peptides having hemostatic activity.
35. The method of claim 24 wherein said substrate comprises from about 0.001 to about 50 percent by weight of said
hemostatic agent.
36. The methodof claim 29 wherein said substrate comprises from about 0.001 to about 1 percent by weight of thrombin
as the hemostatic agent.
37. The methodof claim 29 wherein said substrate comprises from about 0.1 to about 50 percent by weight of fibrinogen
as the hemostatic agent.
13
EP 1 424 086 A1
European Patent
Office
PARTIAL EUROPEAN SEARCH REPORT
Application Number
which under Rule 45 of the European Patent Convention £p 03 25 4088
shall be considered, for the purposes of subsequent
proceedings, as the European search report
DOCUMENTS CONSIDERED TO BE RELEVANT
Category
Citation of document with indication, where appropriate,
of relevant passages
Relevant
to claim
CLASSIFICATION OF THE
APPLICATION (lnt.CI.7)
WO 02 02155 A (C T P CABLE TECHNOLOGY
PROCURE ;RYLT5EV VLADIMIR VALENTIOVICH
(RUJ) 10 January 2002 (2002-01-10)
1-14,
21-35
page
page
page
page
page
1, line 8 - line 12 *
4, line 1 - line 9 *
4, line 33 - page 5, 1 ine
5, line 26 - page 6, 1 ine
8, line 26 - page 9, line
9 *
12 *
15 *
6B 2 314 842 A (JOHNSON & JOHNSON MEDICAL)
14 January 1998 (1998-01-14)
* page 1, line 3 - line 14 *
* page 3, line 18 - page 4, line 34 *
* page 5, line 7 - line 22 *
* page 7, line 9 - line 15 *
US 3 868 955 A (SIRAGUSA JUDITH ANN ET AL)
4 March 1975 (1975-03-04)
* column 2, line 14 - line 49 *
* column 3, line 36 - line 45 *
* column 5, 1 ine 22 - 1 ine 65 *
1-14,
21-35
1-37
A61L15/28
INCOMPLETE SEARCH
The Search Division considers that the present application, or one or more of its claims, does/do
not compfy with the EPC to such an extent that a mean ingfuf search into the state of the art cannot
be carried out, or can only be carried out partially, for these claims
Claims searched completely :
Claims searched incompletely :
Claims not searched :
Reason for the limitation of the search:
see sheet C
TECHNICAL FIELDS
SEARCHED (Irit.CI.7)
A61L
Place of search
THE HAGUE
Date of completion of the search
26 March 2004
Examiner
Menidjel, R
CATEGORY OF CITED DOCUMENTS
X : particularly relevant if taken alone
Y : particularly relevant if combined with another
document of the same category
A : technological background
O : non-written disclosure
P : intermediate document
T : theory or principle underlying the invention
E : earlier patent document, but published on, or
after the filing date
D : document cited in the application
L : document cited for other reasons
& : member of the same patent family, corresponding
document
14
EP 1 424 086 A1
European Patent
Office
INCOMPLETE SEARCH
SHEET C
EP 03 25 4088
Application Number
Although claims 24-37 are directed to a method of treatment of the
human/animal body (Article 52(4) EPC), the search has been carried out
and based on the alleged effects of the compound/composition.
Reason for the limitation of the search (non-patentable invention(s)) :
Article 52 (4) EPC - Method for treatment of the human or animal body by
therapy
15
EP 1 424 086 A1
ANNEX TO THE EUROPEAN SEARCH REPORT
ON EUROPEAN PATENT APPLICATION NO.
EP 03 25 4088
This annex lists the patent family members relating to the patent documents cited in the above-mentioned European search report.
The members are as contained in the European Patent Office EDP file on
The European Patent Office is in no way liable for these particulars which are merely given for the purpose of information.
26-03-2004
Patent document
Publication
Patent family
Publication
cited in search report
date
member(s)
date
WO 0202155 A
10-01-2002
WO
0202155 Al
10-01-2002
AU
EP
5824900 A
1299135 Al
s
cc
O
Li. I
O
w For more details about this annex : see Official Journal of the European Patent Office, No. 12/82
14-01-2002
09-04-2003
GB
2314842
A
14'
-01-
1998
AT
247493
T
15-09-2003
AU
737809
B2
30-08-2001
AU
3269297
A
21-01-1998
BR
9710177
A
18-01-2000
CA
2258990
Al
08-01-1998
CZ
9804251
A3
12-05-1999
DE
69724257
Dl
25-09-2003
EP
1325754
Al
09-07-2003
EP
0918548
Al
02-06-1999
WO
9800180
Al
08-01-1998
JP
2000513258
T
10-10-2000
KR
2000022287
A
25-04-2000
PL
330824
Al
07-06-1999
PT
918548
T
28-11-2003
US
3868955
A
04-
-03-
1975
AT
338980
B
26-09-1977
BE
824345
Al
14-07-1975
DE
2500162
Al
08-07-1976
FR
2297636
Al
13-08-1976
GB
1477571
A
22-06-1977
LU
71650
Al
24-06-1975
NL
7500428
A ,B,
16-07-1976
16